Anti-GFP antibody [RM1064]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GFP antibody [RM1064] (AB316291)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (human embryonic kidney epithelial cell) cells transfected with an empty expression vector containing a myc-His-tag® (upper left and upper right) /293T cells transfected with an GFP expression vector containing an eGFP tag (bottom left and bottom Right) cells labelling GFP with ab316291 at 1/5000 dilution (0.01 ug) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

293T cells transfected with an empty expression vector containing a myc-His-tag® were labeled with anti-Myc antibody crosslinked with Alexa Fluor®647 (670/14BP).And use Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) combined with ab316291.
Isotype are Rabbit monoclonal IgG (ab172730).
293T cells transfected with an GFP expression vector containing an eGFP tag are use Goat Anti-Rabbit IgG (Alexa Fluor® 647, ab150083) combined with ab316291.
Isotype are Rabbit monoclonal IgG (ab172730).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody [RM1064] (AB316291)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling GFP with ab316291 at 1/5000 (0.11 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human colon in ab316291 (Image A) and weak background stainning on the human colon in ab290 (Image B).
The image A is applied with Anti-GFP antibody (ab316291) at 1/5000 dilution and the image B is applied with Anti-GFP antibody (ab290) at 1/5000 dilution.
The section was incubated with ab316291 or ab290 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody [RM1064] (AB316291)

Immunohistochemical analysis of paraffin-embedded (A) 293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a GFP-tag (B) 293T transfected with an empty vector containing an EGFP-myc-tag (C) 293T transfected with an empty vector containing a myc-His-tag® tissue labeling GFP with ab316291 at 1/5000 (0.11 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on 293T transfected with an empty vector containing a GFP-tag cell pellets (A) and 293T transfected with an empty vector containing an EGFP-myc-tag cell pellets (B). No staining on 293T transfected with an empty vector containing a myc-His-tag cell pellets (C).
The upon images are applied with Anti-GFP antibody (ab316291) at 1/5000 dilution and the below images are applied with Anti-GFP antibody (ab290) at 1/5000 dilution.
The section was incubated with ab316291 or ab290 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GFP antibody [RM1064] (AB316291)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) transfected with empty vector containing a EGFP tag. cells labelling GFP with ab316291 at 1/500 (1.096 ug/ml) dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing positive staining in 293T cells transfected with empty vector containing a EGFP tag and 293T cells transfected an GFP expression vector (shown in magenta), and no staining in 293T cells transfected with empty vector containing a myc tag and 293T cells. EGFP and GFP was observed in green. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution.

• ChIP

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ChIP - Anti-GFP antibody [RM1064] (AB316291)

Chromatin was prepared from Hela (human epithelial cell line from cervix adenocarcinoma) transfected with GFP Tagged human CREBBP and Hela transfected with empty vector cells according to the Abcam Dual-X-ChIP protocol*.Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab316291 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody [RM1064] (AB316291)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling GFP with ab316291 at 1/5000 (0.11 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on mouse spleen.
The section was incubated with ab316291 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody [RM1064] (AB316291)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling GFP with ab316291 at 1/5000 (0.11 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on rat spleen.
The section was incubated with ab316291 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• I-ELISA

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Indirect ELISA - Anti-GFP antibody [RM1064] (AB316291)

Indirect ELISA analysis of ab316291 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution.

Antigen : GFP.

Antigen concentration : 1000 ng/ml

• IP

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Immunoprecipitation - Anti-GFP antibody [RM1064] (AB316291)

GFP was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) transfected with a GFP protein expression vector, whole cell lysate with ab316291 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316291 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293T (human embryonic kidney epithelial cell) transfected with a GFP protein expression vector, whole cell lysate
Lane 2 : ab316291 IP in HEK-293T (human embryonic kidney epithelial cell) transfected with a GFP protein expression vector, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of primary antibody in HEK-293T (human embryonic kidney epithelial cell) transfected with a GFP protein expression vector, whole cell lysate.

The left panel is applied with Anti-GFP antibody (ab316291) at 1/1000 dilution and the right panel is applied with Anti-GFP antibody (ab290) at 1/1000 dilution.

All lanes:

Immunoprecipitation - Anti-GFP antibody [RM1064] (ab316291) at 1/30 dilution

All lanes:

HEK-293T (human embryonic kidney epithelial cell) transfected with a GFP protein expression vector, whole cell lysate with 5% NFDM/TBST

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 1s

• WB

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Western blot - Anti-GFP antibody [RM1064] (AB316291)

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

Exposure time : Lanes 1-2 : 8 seconds; Lane 3 : 37 seconds

All lanes:

Western blot - Anti-GFP antibody [RM1064] (ab316291) at 1/5000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 10 µg with 5% NFDM/TBST

Lane 2:

293T transfected with an empty vector containing an EGFP-myc-tag, whole cell lysate at 10 µg with 5% NFDM/TBST

Lane 3:

293T transfected with an empty vector containing a GFP-tag, whole cell lysate at 20 µg with 5% NFDM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 27 kDa,15 kDa

false